This invention relates to a detection of a partly complementary nucleic acid fragment. The detection of a partly complementary nucleic acid fragment is of value in the gene analyses such as analysis of gene polymorphism and analysis of variation of abnormal gene.
The gene analysis is recently paid an attention in developing gene technology.
As a representative gene analysis method for detecting variation of gene fragments, SSCP (single-stranded conformation polymorphism) is known. The procedure of SSCP for differentiating an abnormal gene fragment from a normal gene fragment is illustrated in FIG. 1 attached to the specification. Each of double stranded normal gene fragments (11a) and double stranded abnormal gene fragments (11b, which is different from the normal gene fragment 11a in the base unit positioned in the crossed place) is treated (31) to give a set of two single stranded gene fragments (21a, 21b), as illustrated in FIG. 1-(A). Both of the single stranded gene fragments (21a, 21b) are subjected to electrophoresis on a polyacrylamide gel. Each gene fragment has a specific high-order structure differing from each other. Accordingly, each of the single stranded gene fragments (21a, 21b) moves differently from each other in the electrophoresis, as illustrated in FIG. 1-(B). The detection of the movement of the gene fragment gives an information of variation of gene fragments.
The SSCP meth, however, sometimes fails to differentiate an abnormal gene fragment from a normal gene fragment, because a certain abnormal gene fragment moves similarly to the corresponding normal gene fragment. Particularly, if the variation of base takes place in the vicinity of a gene fragment, it is likely that the abnormal gene fragment takes almost the same high-order structure as that of the corresponding normal gene fragment, and hence that there is caused almost no difference of movement in the electrophoresis.
The variation of gene fragment from a single stranded normal gene fragment to a single stranded abnormal gene fragment can be also detected by way of hybridization. In the hybridization, a hybrid structure is formed differently between the normal gene fragment and the abnormal gene fragment.
Japanese Patent PCT Publication 9-501561 describes detection of a double-stranded nucleic acid hybrid having a specific structure by means of a fluorescent microscope.
It is also known that different double-stranded nucleic acid fragment hybrids are detected by temperature graduation gel electrophoresis.
It is known that there are three types of hybrid structures, namely,
a full-match structure in which two single stranded DNA fragments are hybridized to form a complete double-stranded DNA fragment;
a mis-match structure in which two single stranded DNA fragments are hybridized to form a double-stranded DNA fragment having one uncoupled base unit, which is generally observed in the case that a normal DNA fragment is hybridized with an abnormal DNA fragment which differs from the normal DNA fragment in one base unit; and
a bulge structure in which two single stranded DNA fragments are hybridized to form a double-stranded DNA fragment having two or more uncoupled base units.
Japanese Patent Provisional Publication No. 9-288080 describes a method for detecting a double stranded DNA fragment of a full-match structure by way of an electrochemical detection method. In this method, a sample DNA fragment is electrochemically detected using a DNA probe which is complementary to the sample DNA fragment in the presence of an electrochemically active thread intercalator.
P. E. Nielsen et al., Science, 254, 1497-1500(1991) and P. E. Nielsen et al., Biochemistry, 36, pp.5072-5077 (1997) describe PNA (Peptide Nucleic Acid or Polyamide Nucleic Acid) which has no negative charge and functions in the same manner as DNA does. PNA has a polyamide skeleton of N-(2-aminoethyl)glycine units and has neither glucose units nor phosphate groups. A representative PNA as well as a representative DNA are illustrated below: 
Since PNA is electrically neutral and is not charged in the absence of an electrolytic salt, PNA is able to hybridize with a complementary nucleic acid fragment to form a hybrid which is more stable than hybrid given by a DNA prove and its complementary nucleic acid fragment (Preprint of the 74th Spring Conference of Japan Chemical Society, pp. 1287, reported by Naomi Sugimoto).
Japanese Patent Provisional Publication No.11-332595 describes a PNA probe fixed on a electroconductive substrate at its one end and a detection method utilizing the PNA probe. The PNA probe is fixed onto the electroconductive substrate by the avidin-biotin method.
The aforementioned P. E. Nielsen et al, Science, 254, 1497-1500(1991) also describes a PNA probe labelled with isotope element and a detection method of a complementary nucleic acid fragment.
Since the PNA probe shows no electric repulsion to a target nucleic acid fragment in a sample liquid, an improved high detection sensitivity is expected.
It is an object of the present invention to provide a method of analyzing a nucleic acid fragment sample to judge whether the nucleic acid fragment sample is uncomplementary, partly complementary, or complementary to a DNA fragment or PNA fragment in its specific base sequence. This analyzing method is of value in the gene analysis such as analysis of gene polymorphism or analysis of variation of abnormal gene.
The present invention resides in a method of analyzing a nucleic acid fragment sample to judge whether the nucleic acid fragment sample is uncomplementary, partly complementary or complementary to a DNA fragment in its specific base sequence, which comprises the steps of:
bringing an aqueous solution of the nucleic acid fragment sample into contact with a DNA chip comprising an electroconductive substrate and the DNA fragment fixed onto the substrate in the presence of an electrochemical thread intercalator;
measuring an electric current flowing from or to the electroconductive substrate along the DNA fragment under application of a potential to the substrate; and
comparing the electric current measured above with a referential electric current which is prepared employing a combination of a DNA chip equivalent to the above-mentioned DNA chip, the electrochemical thread intercalator, and an aqueous solution of a nucleic acid fragment which is complementary to the DNA fragment of the DNA chip.
The invention also resides in a method of analyzing a nucleic acid fragment sample to judge whether the nucleic acid fragment sample is uncomplementary, partly complementary or complementary to a PNA fragment in its specific base sequence, which comprises the steps of:
bringing an aqueous solution of the nucleic acid fragment sample into contact with a PNA chip comprising an electroconductive substrate and the PNA fragment fixed onto the substrate in the presence of an electrochemical thread intercalator;
measuring an electric current flowing from or to the electroconductive substrate along the PNA fragment under application of a potential to the substrate; and
comparing the electric current measured above with a referential electric current which is prepared employing a combination of a PNA chip equivalent to the above-mentioned PNA chip, the electrochemical thread intercalator, and an aqueous solution of a nucleic acid fragment which is complementary to the PNA fragment of the PNA chip.
The above-defined detection methods of the invention preferably further comprises the steps of:
bringing an aqueous solution not containing the nucleic acid fragment sample into contact with a DNA chip (or a PNA chip) equivalent of the DNA chip (or the PNA chip) in the presence of the electrochemical thread intercalator;
measuring an electric current flowing from or to the electroconductive substrate along the DNA fragment (pr PNA fragment) under application of a potential to the substrate so as to obtain a background electric current; and
comparing the background electric current with the electric current which is measured employing the aqueous solution containing the nucleic acid fragment sample.
In the present detection method, the specific base sequence of the DNA fragment (or PNA fragment) is preferably predetermined. The DNA chip (or PNA chip) employed in the detection method of the invention preferably has a plurality of short chain spacer molecules having a hydrophilic moiety at each one end which are fixed at each another end onto a surface area of the electroconductive substrate having no DNA fragments (or no PNA fragments) thereon. The electrochemical thread intercalator preferably is a ferrocene-containing thread intercalator having an oxidative-reductive activity.
The measurement of the electric current in each step in the detection method of the invention is preferably conducted by differential pulse voltamography.